The present invention pertains to novel strains of Pseudomonas corrugata and uses of the strains in biological control against soil-borne pathogens of plants. Also encompassed by this invention is a method of identifying and selecting desiccation tolerant strains of bacteria and a method of producing the desiccated formulations of the identified strains.
Bacterial and fungal pathogens can result in significant yield losses or decrease the market quality of agricultural crops. Under favorable conditions, soil resident fungal pathogens may decrease seedling emergence, cause root rots, or reduce plant vigor. Many fungal pathogens produce spores that aid both dispersal and survival of the fungus. Bacteria, are single-celled organisms that generally do not produce spores. Many bacterial pathogens depend on host material for survival. Other bacterial pathogens may be able to survive in the soil as saprophytes.
For both types of pathogens, entry into plant tissue is required for the production of disease symptoms. Prevention of pathogen entry would significantly decrease the likelihood of disease. Chemical seed treatment with fungicides can be used to provide economical protection against some soil-borne fungal pathogens. Antagonistic bacteria may also be used to provide biological control protection against undesirable pathogens. However, biological control bacteria must be able to survive drying to be readily employed as a seed treatment. Additionally, compatibility with fungicides would enable the combination of biological controls with chemicals to achieve disease control.
Crops are susceptible to a variety of different pathogens. For example, peas are susceptible to a variety of root-infecting fungi (Aphanomyces euteiches, Fusarium oxysporum f. sp. pisi, F.solani f. sp. pisi, and Sclerotinia sclerotiorum). Several Pythium species (P. debaryanum, P. ultimum, P. splendens, P. aphanidermatum, and P. irregulare) are known to cause damping-off and root rot in peas. Many of these same pathogens are pathogenic on potato. Peas and potatoes are also known hosts for the bacterial leaf spot pathogen, Pseudomonas pisi, and the bacterial ring rot pathogen, Clavibacter michiganensis subsp. sepedonicus, respectively.
Aphanomyces euteiches (the causal agent of Aphanomyces root rot) by far is the most serious disease threat to pea production. The fungus produces sporangia that release many motile zoospores that xe2x80x9cswimxe2x80x9d to susceptible root tissue. Infection occurs after zoospores encyst and form germ tubes that penetrate the epidermis of young roots. The fungus also produces thick-walled oospores that are the primary overwintering structures. These spores may germinate directly or form sporangia in the presence of young pea plants. Oospores of A. euteiches have been know to survive for as long as 20 years in the soil in the absence of any crops. At present no chemical control is available and resistant cultivars are lacking. Cool moist conditions that normally exist as the seeds germinate are optimal conditions for this pathogen. Thus, alternative methods are needed to manage this disease.
Ring rot disease caused by Clavibacter michiganensis subsp. sepedonicus is a major concern in potato seed production. The bacterium""s ability to survive on tools, equipment, dried infected plant material, volunteer plants, insects and alternative hosts has made eradication impossible. Furthermore, the pathogen may escape detection during field surveys and seed tests as symptoms are not always expressed in infected plants and tubers. Disease control ultimately rests on the accurate and sensitive detection of the bacterial ring rot pathogen. While disease incidence is low, presence of the disease results in rejection of the crop and loss of the field for five to seven years. Chemical control and resistant cultivars are not available for control of this disease.
Silver scurf is a post-harvest disease of potato tubers. Infection occurs in the field from inoculum carried on seed potatoes. In storage bins, lesions on the potato surface appear. This results in a significant decrease in quality as well as moisture loss by tubers. Recently strains have developed that are resistant to current fungicides.
Several different bacteria have been used as biological control agents for fungal diseases (Weller, 1988). The majority of antagonistic bacteria isolated from soil that displayed antagonistic properties were fluorescent pseudomonads (de la Cruz et al., 1992; Kloepper, 1983; Kloepper and Schroth, 1978; Liu et al., 1995; Lorang et al., 1995; McLoughlin et al., 1992; Rodriguez and Pfender, 1997; Thompson et al., 1996; Vidhyasekaran and Muthamilan, 1995.; Weller, 1988; Xu and Gross, 1986; Zhou and Paulitz, 1993). Other studies have shown that Gram positive bacteria such as Streptomyces and Bacillus may have some promise (Broadbent et al., 1977; Crawford, 1996; Liu et al., 1995; Lorang et al., 1995). However, phytopathogenic bacteria have not been extensively evaluated as biological control agents. Burkholderia cepacia (Pseudomonas cepacia) has been used in such studies (Parke, 1992; Parke et al., 1991). In some instances, B. cepacia strains used were obtained from soil and were not pathogenic on plants (Novitski et al., 1993).
Pseudomonas corrugata was first reported as a pathogen of tomatoes causing a necrosis of the pith (Scarlett et al., 1978). This bacterium has also been reported as a pathogen of rice (Cottyn et al., 1996) and pepper (Lopez et al., 1994). P. corrugata may be more widely present in agricultural soils (Schisler and Slininger, 1994; Scortichini, 1989) and has been isolated from alfalfa (Lukezic, 1979) and wheat (Ryder and Rovira, 1993) roots.
Since the host range of P. corrugata appears to be limited to tomato, pepper and possibly rice, wild-type isolates could be used on non-susceptible crops. Soil-borne isolates were recovered as antagonists of the potato scab pathogen, Streptomyces scabies (Schisler and Slininger, 1994). Other isolates have been found on wheat roots and have been used to suppress take-all disease of wheat (Ryder and Rovira, 1993). Additionally, suppression of Pythium root rot on cucumber was obtained by root treatment with P. corrugata (Zhou and Paulitz, 1993).
What is still needed is alternative biological control methods and agents against plant diseases, particularly for diseases where chemical control agents or resistant cultivars are unavailable or undesirable.
The present invention pertains to novel strains of Pseudomonas corrugata and the use of the strains in biological control against soil-borne pathogens of plants. A number of these strains are shown to be effective in reducing or controlling soil-borne pathogens of plants, including, but not limited to peas, potatoes and wheat. In particular the present invention pertains to 3 strains of Pseudomonas corrugata herein referred to as 0782-6, 0683-32 and 1090-11. P. corrugata isolates 0782-6 and 0683-32 have been identified as having the strongest antimicrobial activity to a variety of plants. Isolate 1090-11 showed little or no antifungal activity.
The P. corrugata strains may be formulated with a variety of delivery media to produce bacterial inocula, that may be used as either a dry powder or liquid suspension. The formulations may be introduced into the soil or may be applied to seeds, plant surfaces or portions of plants, such as roots, stems, leaves, and fruit, or plant parts may be dipped into the formulations, to treat the plants.
Also encompassed by this invention is a method of identifying and selecting desiccation tolerant strains of bacteria and a method of producing the desiccated formulations of the identified strains. The method comprises, in general, culturing the bacteria in a minimal nutrient broth medium containing a single carbon source (MMSC) for a minimum of approximately 24 hours. After the culturing of the bacteria for 24 hours, samples of the MMSC cultures are dried. The dried samples are then rehydrated, and plated on a complete nutrient substrate. The next step comprises identifying which strains have a high rate of survival by comparing the rate of survival of the rehydrated samples to the rate of survival of the bacteria cultured in the same minimal nutrient broth and selecting the strains having a high rate of survival. This process is repeated until no increase in survival is observed. In the case of the novel strains of Pseudomonas corrugata of the present invention, the composition comprising the desiccated strains and delivery medium has a long shelf life and is suitable for delivering the bacteria to plants for effective control of plant pathogens.